CN115282135B - Application of enantiomer-kaurane diterpenoid DKA in preparation of anti-tumor metastasis drugs or inhibitors - Google Patents
Application of enantiomer-kaurane diterpenoid DKA in preparation of anti-tumor metastasis drugs or inhibitors Download PDFInfo
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Abstract
The invention discloses an application of an enantiomer-kaurane diterpenoid DKA in preparing anti-tumor invasion and metastasis medicaments or inhibitors, wherein the enantiomer-kaurane diterpenoid DKA is separated from herba siegesbeckiae, and the enantiomer-kaurane diterpenoid DKA has no cytotoxic activity and can remarkably inhibit cell migration and MDA-MB-231 breast cancer cell lung metastasis through transwell tests, scratch tests, mouse breast cancer lung metastasis and other tests. Has remarkable anti-tumor metastasis effect, and can be used for preparing medicines or inhibitors for resisting tumor invasion and metastasis.
Description
Technical Field
The invention relates to an application of an enantiomer-kaurane diterpenoid DKA in preparation of an anti-tumor metastasis drug or inhibitor, belonging to the technical field of biological pharmacy.
Background
Breast cancer is one of the most common malignant tumors among women worldwide, severely threatening the health of the women. Breast cancer metastasis is a major cause of its clinical failure. The treatment process of early breast cancer (stage I and stage II) generally comprises tumor excision operation, and then chemotherapy or radiotherapy, so that the aim of curing most breast cancer patients can be fulfilled. However, the chemotherapeutic agent anthracyclines have strong cardiotoxicity, such as can cause cardiomyopathy and cardiogenic syncope. Epidemiological studies have shown that 50% of breast cancer patients develop heart lesions, 40% of patients develop arrhythmia, and 5% of patients develop heart failure after the end of anthracycline chemotherapy for 10-20 years. Furthermore, the survival rate of patients with breast cancer metastasis or advanced stage at the end of anthracycline chemotherapy for 10 years is only 22%. The existing clinically applied medicines cannot fundamentally control the occurrence and development of diseases, especially hormone medicines, and are difficult to keep taking medicines for a long time due to the wide side effects of the hormone medicines. Therefore, the finding of lead compounds with good curative effect and few side effects from traditional Chinese medicines with wide clinical application is an important way for developing medicaments for resisting breast cancer tumor metastasis.
Siegesbeckiae herba is annual herb of Siegesbeckiae herba (Siegesbeckia) belonging to Compositae (Asteraceae). The dry overground parts of 3 plants of the asteraceae plants siegesbeckia (Siegesbeckia orientalis l.), siegesbeckia glandula (Siegesbeckia pubescens m.) and siegesbeckia pubescens (Siegesbeckia glabrescent m.) are collected in the 2015 edition of the chinese pharmacopoeia and used as traditional Chinese medicine siegesbeckia orientalis. Modern pharmacological experiments show that the siegesbeckia herb extract has anti-inflammatory, antiallergic, antithrombotic, antitumor, antihistaminic activities and the like, and diterpene is the main active ingredient of siegesbeckia herb. The diterpenoid compounds separated from herba siegesbeckiae are enantiomer-16, 17-dihydroxykauran-19-carboxylic acid (DKA), and pharmacological activity researches show that the diterpenoid compounds have better antithrombotic activity, the application of the compounds is narrower at present, and new applications of the compounds in other fields are urgently needed to be developed.
Disclosure of Invention
The invention aims to: aiming at the technical problems existing in the prior art, the invention aims to provide the application of an enantiomer-kaurane diterpenoid compound DKA (enantiomer-16β, 17-dihydroxykaurane-19-carboxylic acid) in preparing anti-tumor invasion and metastasis medicaments or inhibitors.
The technical scheme is as follows: the invention relates to an application of an enantiomer-kaurane diterpenoid compound DKA in preparing anti-tumor invasion and metastasis medicaments.
The invention also comprises application of the enantiomer-kaurane diterpenoid DKA in preparing an anti-tumor invasion and metastasis inhibitor.
Wherein the tumor is a breast tumor.
The preparation method of the enantiomer-kaurane diterpenoid DKA comprises the following steps:
(1) Reflux-extracting the dry part of Siegesbeckiae herba with ethanol for several times, filtering, and collecting filtrate;
(2) Concentrating the filtrate under reduced pressure to obtain soft extract;
(3) Dispersing the thick extract into water, stirring uniformly, extracting with ethyl acetate for multiple times, mixing ethyl acetate solutions, concentrating the ethyl acetate solutions under reduced pressure to obtain ethyl acetate part extract;
(4) Subjecting the ethyl acetate extract to silica gel column chromatography, eluting with chloroform-methanol system, collecting chloroform-methanol eluate, eluting with silica gel column chromatography, eluting with petroleum ether-ethyl acetate system for 15-20 column volumes until white powder or crystal is separated out, collecting the eluate, concentrating the eluate until a large amount of white powder is separated out, filtering, and washing with petroleum ether-ethyl acetate to obtain purified product of enantiomer-kaurane diterpenoid DKA.
In the step (1), the number of times is more than 3 and each time is 1.5 hours.
Wherein, in the step (2), the filtrate is concentrated under reduced pressure until the density is 1.15-1.20g/ml, and no obvious ethanol smell exists.
Wherein in the step (3), the mass ratio of the thick extract to water is 1:20, and the volume ratio of the ethyl acetate to the water solution is 1.5-2:1 when the ethyl acetate is used for extraction, and the extraction is performed for more than three times.
In the step (4), the chloroform-methanol eluting part is eluted by using a chloroform-methanol system when silica gel column chromatography is carried out, wherein the volume ratio of chloroform to methanol is 30:1-10:1, and specifically, 30:1, 20:1 and 10:1 respectively.
Wherein in the step (4), the chloroform-methanol elution part is the liquid collected when the volume ratio of chloroform to methanol is 30:1.
In the step (4), the chloroform methanol elution part is eluted by using a petroleum ether-ethyl acetate system when subjected to silica gel column chromatography, the volume ratio of petroleum ether to ethyl acetate is 6:1, and the white powder is washed by using the petroleum ether-ethyl acetate system with the volume ratio of 6:1-10:1.
The invention provides a rapid preparation method of an enantiomer-kaurane diterpenoid compound DKA, and the compound has obvious effect of resisting tumor metastasis of breast cancer through a transwell test, a scratch test and a mouse breast cancer lung metastasis test.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The MDA-MB-231 cell viability of 7 concentrations measured by the MTT method shows that the enantiomer-kaurane diterpenoid DKA has no cytotoxic activity.
(2) Compared with a Control group, the enantiomer-kaurane diterpenoid DKA can obviously inhibit cell migration through a Tanswell laboratory experiment and a scratch experiment.
(3) Compared with the Control group, the weight of each group of mice is not obviously different, and the enantiomer-kaurane diterpenoid DKA can obviously inhibit MDA-MB-231 breast cancer cell lung metastasis.
Drawings
Fig. 1 is a flow chart of the preparation of an enantio-kaurane diterpenoid DKA;
fig. 2 is a nuclear magnetic hydrogen spectrum of an enantiomer-kaurane diterpenoid DKA;
FIG. 3 is a nuclear magnetic carbon spectrum of an enantiomer-kaurane diterpenoid DKA;
FIG. 4 is a graph of MTT assay results;
FIG. 5 is a graph showing the inhibition of MBA-MD-231 metastasis by the enantiomer-kaurane diterpenoid DKA;
FIG. 6 is a graph of scratch recovery from 0-72 hours;
FIG. 7 is a graph of scratch healing rate;
fig. 8 is a photograph of lung of mice injected with different concentrations of enantio-kaurane diterpenoid DKA for 4 weeks;
fig. 9 is a graph of body weight of mice injected with different concentrations of enantio-kaurane diterpenoid DKA for 4 weeks;
FIG. 10 is a graph of HE staining of lung tissue;
FIG. 11 is a graph of intrapulmonary transformation rates.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1
1. Preparation of enantiomer-kaurane diterpenoid DKA:
the preparation flow is shown in figure 1:
(1) Reflux-extracting 5kg of Siegesbeckiae herba with 40L ethanol for 3 times each for 1.5 hr, filtering, and collecting filtrate;
(2) Concentrating the filtrate under reduced pressure to obtain soft extract with density of 1.15-1.20g/ml without obvious ethanol taste;
(3) Dispersing the thick extract and water into water according to the proportion of 1:20, stirring uniformly to obtain water solution, extracting with ethyl acetate, wherein the volume ratio of the ethyl acetate to the water solution is 1.5-2:1, extracting for three times, combining ethyl acetate solutions, and concentrating under reduced pressure to obtain ethyl acetate part extract;
(4) Subjecting the ethyl acetate part extract to silica gel column chromatography, eluting with chloroform-methanol system (volume ratio of chloroform-methanol is 30:1, 20:1, 10:1), collecting chloroform-methanol 30:1 eluate, subjecting the collected chloroform-methanol eluate to silica gel column chromatography, eluting with petroleum ether-ethyl acetate system (volume ratio of petroleum ether-ethyl acetate is 6:1), eluting for 15-20 column volumes until white powder or crystallization is present, collecting the part eluate, concentrating the collected eluate until a large amount of white powder is present, precipitating, filtering, and washing with petroleum ether-ethyl acetate system (volume ratio of petroleum ether-ethyl acetate is 6:1) to obtain purified product of enantiomer-kaurane diterpenoid DKA.
2. The structure of purified enantiomer-kaurane diterpenoid DKA was confirmed:
mass spectrometry of white powder of purified enantiomer-kaurane diterpenoid DKA, positive ESI-MS m/z 359.5[ M+Na ]] + Thus, the molecular weight was 336, molecular formula C 20 H 32 O 4 。
The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum analysis are carried out on the purified matter of the enantiomer-kaurane diterpenoid DKA, the results are shown in figures 2-3, figure 2 is the nuclear magnetic hydrogen spectrum of the enantiomer-kaurane diterpenoid DKA, and as can be seen from figure 2, 1 H NMR(Pyridine-d 5 400 MHz) spectrum has 2 monomodal methyl peak signals 1.33 (3H, s, me-18), 1.17 (3H, s, me-20), the low field shows no double bond hydrogen signals and 2 hydrogen signals on the oxygen-attached carbon: 4.10 (1 h, d, j=10.8 hz, ha-17), 4.03 (1 h, d, j=10.8Hz,Hb-17)。
FIG. 3 shows a nuclear magnetic carbon spectrum of an enantiomer-kaurane diterpenoid DKA, as can be seen from FIG. 3, 13 C NMR(Pyridine-d 5 100 MHz) gives a 20 carbon atom signal: C 41.0(C-1),19.8(C-2),38.7(C-3),43.9(C-4),57.0(C-5),22.9(C-6),42.7(C-7),44.9(C-8),56.3(C-9),40.0(C-10),19.0(C-11),26.2(C-12),45.9(C-13),37.8(C-14),53.8(C-15),81.6(C-16),66.5(C-17),29.3(C-18),180.1(C-19),16.0(C-20)。
by the thin layer identification and the nuclear magnetic data of the compound, the compound is determined to be enantiomer-16, 17-dihydroxykaurane-19-carboxylic acid, and the structural formula is as follows:
3. MTT experiment:
MBA-MD-231 cells (obtained from American Tissue Culture Collection) in logarithmic growth phase, adjusted to 5000 cells/well, inoculated into 96-well plates, and placed at 37deg.C, 5% CO 2 Culturing under conditions until cells are 90% confluent, incubating with serum-free Leibovitz's L-15 medium for 2h to synchronize the cells, treating the cells in groups of 0. Mu.M, 0.1. Mu.M, 0.3. Mu.M, 1. Mu.M, 3. Mu.M, 10. Mu.M, 30. Mu.M, 100. Mu.M for 72h, carefully pipetting the supernatant, adding 90. Mu.l of a medium containing penicillin (100U/mL final concentration), streptomycin (100. Mu.g/mL final concentration), 10% FBS in Leibovitz's L-15, and adding 10. Mu.l of MTT solution at 37℃and 5% CO 2 Culturing under the condition for 4 hours, sucking the supernatant after the incubation, adding 110 mu l Formazan dissolving solution into each hole, and shaking at low speed on a shaking table for 10 minutes to fully dissolve the crystals. Absorbance was measured at 490nm for each well in an enzyme-linked immunosorbent assay. The results are shown in FIG. 4. Fig. 4 is a graph showing the results of the MTT experiment, and it can be seen from fig. 4 that there is no cytotoxic activity of the enantiomer-kaurane diterpenoid DKA.
4. The Tanswell cell method detects the influence of the enantiomer-kaurane diterpenoid DKA on the transfer capacity of MBA-MD-231 cells, and evaluates the inhibition activity of the enantiomer-kaurane diterpenoid DKA on the transfer capacity of MBA-MD-231:
(1) MBA-MD-231 cells were plated onto 6-well plates and placed in 37,5% CO 2 Is cultured in incubator for 24 hours to adhere to the incubator, DKA samples (DKA aqueous solution containing DMSO) with different concentrations (1. Mu.M, 5. Mu.M, 25. Mu.M) are added, and the incubator is placed at 37 ℃ with 5% CO 2 Incubating in the incubator for 24 hours.
(2) After incubation, cells from each well were digested with 1mL of 0.25% pancreatin, stopped with 1mL of 10% FBS-containing medium, centrifuged at 1300r/min for 5min, the supernatant was decanted, mixed with 1mL of 0.1% BM, centrifuged at 1300r/min for 5min, counted, and cell density at 200-300. Mu.L was adjusted to a final concentration of 5X 10 5 The cell suspension is placed in an incubator for standby.
(3) Preparing chemokine EGF on ice, adding 30 mu L of each hole into the lower chamber of the chemotactic cell; and (3) cutting the left upper corner of the coated polycarbonate film, spreading the polished surface downwards on the lower chamber, placing a rubber pad, and fixing the upper chamber.
(4) The cell suspension was added to the upper chamber at 50. Mu.L per well, 3 multiplex wells per sample concentration, and then placed in CO 2 After 3.5h incubation in incubator, the lower chamber was removed, the non-chemotactic cells were scraped off, fixation and staining were performed with three-step staining reagent, and after staining, the membrane was fixed with paraffin oil and counted under microscope. This experiment selected 2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one (LY 294002) as the positive control. The experimental results of the inhibitory activity of the enantiomer-kaurane diterpenoid DKA on MBA-MD-231 metastasis are shown in Table 1 and FIG. 5.
TABLE 1 inhibition of MBA-MD-231 metastasis by different concentrations of DKA
Experimental results show that the enantiomer-kaurane diterpenoid DKA is in IC 50 At a value of 1.96 (. Mu.M), there was 50% inhibition of breast cancer tumor cell metastasis, which was slightly weaker than the positive control LY294002 (2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one hydrochloride).
5. Scratch experiment:
(1) Scratch experiment: after culturing each group of MBA-MD-231 cells in DKA groups of 1. Mu.M, 5. Mu.M and 25. Mu.M for 72 hours, the medium was discarded, 1ml of a 1. Mu.g/ml mitomycin solution was added, and the mixture was allowed to stand in a conventional incubator at 37℃for 1 hour; the liquid in the wells was aspirated, 1ml of PBS solution (1X, pH 7.4) was added, and 10. Mu.l of the gun head was marked with vertical lines perpendicular to the plate surface and the back side horizontal line, and the liquid in the wells was discarded and gently rinsed 3 times with PBS solution. Mu.l of Leibovitz's L-15 medium each containing 1% FBS was added, and the mixture was placed in an incubator at 37℃for conventional culture.
(2) Shooting: and shooting under a microscope after the scratch for 0h and 72h, recording shooting coordinates, and ensuring the consistency of shooting parts each time by marking the parallel line positions on the back side surface.
(3) Analysis: each group of cell scratch areas was analyzed with ImageJ software, the percent scratch healing was calculated and the experiment was repeated three times. The results are shown in FIGS. 6-7. Fig. 6 is a graph showing scratch recovery from 0 to 72 hours, and as can be seen from fig. 6, DKA of 1 μm, 5 μm, 25 μm can suppress recovery from scratches. Fig. 7 is a graph showing the rate of scratch healing, and as can be seen from fig. 7, DKA of 1 μm, 5 μm, 25 μm can significantly inhibit scratch healing, with a significant difference from the control group.
6. MBA-MD-231 mice lung metastasis inhibition experiment:
taking MBA-MD-231 cells in logarithmic growth phase, preparing cell suspension with the concentration of 2X 10-6 cell/ml, inoculating 0.2 ml/mouse to nude mice through tail vein, and establishing MBA-MD-231 mouse lung transfer model.
Control group: MBA-MD-231 cells were seeded and 100. Mu.l DMSO was injected tail vein every 2 days for 4 weeks.
Low concentration group of enantiomer-kaurane diterpenoid DKA (2.5 mg/kg): MBA-MD-231 cells were inoculated and 100. Mu.l of the enantiomer-kaurane diterpenoid DKA (final concentration 2.5 mg/kg) was injected intravenously every 2 days for 4 weeks.
Concentration group in DKA (5 mg/kg): MBA-MD-231 cells were inoculated and 100. Mu.l of the enantiomer-kaurane diterpenoid compound DKA (final concentration 5 mg/kg) was injected intravenously every 2 days for 4 weeks.
DKA high concentration group (10 mg/kg): MBA-MD-231 cells were seeded and 100. Mu.l DKA (final concentration 10 mg/kg) was injected intravenously every 2 days for 4 weeks.
Mice were checked for body weight once a week. After 4 weeks of inoculation, all mice were sacrificed by carbon dioxide method, lungs were removed, irrelevant tissues were carefully removed, washed 2-3 times with D-Hanks' solution, blood was washed off, and water was drained for preservation. The results are shown in FIGS. 8-9. Fig. 8 is a photograph of the lungs of mice injected with different concentrations of the enantiose-kaurane diterpenoid compound DKA for 4 weeks, and as can be seen from fig. 8, DKA (2.5 mg/kg,5mg/kg and 10 mg/kg) can significantly reduce the number of macroscopic metastasis nodules on the whole lung surface compared to the Control group. Fig. 9 is a graph of body weight of mice injected with different concentrations of the enantios-kaurane diterpenoid compound DKA for 4 weeks, and as can be seen from fig. 9, there is no significant difference in body weight among the mice in each group.
7. HE experiment:
(1) Cutting the fixed lung tissue into slices with the thickness of 4 mu m, and placing the slices in an oven for 1h;
(2) Performing conventional xylene dewaxing on the dried paraffin sections, hydrating with descending gradient ethanol, and washing with distilled water;
(3) Adding hematoxylin for dyeing for 10-30 min, and then washing off hematoxylin dye liquid by running water;
(4) Fading 1% ethanol hydrochloride until the slice turns red and the color is lighter, and putting the slice into running water to restore blue;
(5) Eosin staining for 1min, washing with running water;
(6) Dehydrating and drying the slices by using gradient alcohol, and sealing the slices by using neutral resin, wherein the xylene is transparent;
(7) Randomly selecting a field of view and taking a photograph using a microscope (400×);
(8) Observing and calculating the number of whole lung metastasis of each mouse;
(9) Statistical differences between data analysis test data sets were examined using one-way ANOVA and Tukey's, with P values less than 0.05 considered significant differences. The results are shown in FIGS. 10-11.
Fig. 10 is a graph of HE staining of lung tissue, and fig. 11 is a graph of the transformation rate of intrapulmonary disease, as can be seen from fig. 10-11, DKA (2.5 mg/kg,5mg/kg and 10 mg/kg) can significantly inhibit lung metastasis of MDA-MB-231 breast cancer cells compared to the Control group.
Claims (8)
1. Application of enantiomer-kaurane diterpenoid compound DKA in preparation of medicaments for resisting invasion and metastasis of breast tumor is provided, wherein the enantiomer-kaurane diterpenoid compound DKA has the following structural formula:
。
2. use of an enantiomer-kaurane diterpenoid compound DKA in the preparation of an inhibitor for inhibiting invasion and metastasis of breast tumor, wherein the enantiomer-kaurane diterpenoid compound DKA has the following structural formula:
。
3. the use according to claim 1 or 2, characterized in that the process for the preparation of the enantios-kaurane diterpenoid DKA comprises the following steps:
(1) Reflux-extracting the dry part of Siegesbeckiae herba with ethanol for several times, filtering, and collecting filtrate;
(2) Concentrating the filtrate under reduced pressure to obtain soft extract;
(3) Dispersing the thick extract into water, stirring uniformly, extracting with ethyl acetate for multiple times, mixing ethyl acetate solutions, concentrating the ethyl acetate solutions under reduced pressure to obtain ethyl acetate part extract;
(4) Subjecting the ethyl acetate extract to silica gel column chromatography, eluting with chloroform-methanol system, collecting chloroform-methanol eluate, eluting with silica gel column chromatography, eluting with petroleum ether-ethyl acetate system for 15-20 column volumes until white powder or crystal is separated out, collecting the eluate, concentrating the eluate until a large amount of white powder is separated out, filtering, and washing with petroleum ether-ethyl acetate to obtain purified product of enantiomer-kaurane diterpenoid DKA.
4. The use according to claim 3, wherein in step (1), the number of times is more than 3 and each time is 1.5 hours.
5. The method according to claim 3, wherein in step (2), the filtrate is concentrated under reduced pressure to a density of 1.15-1.20g/ml without significant ethanol smell.
6. The use according to claim 3, wherein in step (3), the mass ratio of the thick extract to water is 1:20, and the volume ratio of ethyl acetate to water solution is 1.5-2:1 when ethyl acetate is used for extraction, and the extraction is performed for more than three times.
7. The method according to claim 3, wherein in step (4), the chloroform-methanol eluate (chloroform-methanol) is collected at a volume ratio of chloroform to methanol of 30:1.
8. The use according to claim 3, wherein in step (4), the chloroform methanol eluted part is eluted by using a petroleum ether-ethyl acetate system when subjected to silica gel column chromatography, the volume ratio of petroleum ether-ethyl acetate is 6:1, and the white powder is washed by using a petroleum ether-ethyl acetate system with a volume ratio of 6:1-10:1.
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| CN1594267A (en) * | 2004-07-02 | 2005-03-16 | 南京中医药大学 | Diterpenoid monomer and its preparation method and application for preparing anti-cancer drugs |
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